Mammalian defensins are cationic peptides that possess antimicrobial activities against diverse pathogens and also contribute to immune responses by modulating the effector cells of adaptive immunity. Defensins are classified in three structural classes: ?-, ?- and ?-defensins which are produced in their pre-proforms and then converted to the mature form. Human neutrophils contain myeloid ?-defensins (human neutrophil peptides;HNPs) in their azurophil granules wherein they exist exclusively their mature forms. However, during the biosynthesis of these molecules, a fraction of the precursors are secreted from the cell. Further, recent studies demonstrate that HNP-biosynthesis in monocytes and NK cells is inducible revealing that as yet uncharacterized biosynthetic regulatory processes determine the expression of mature defensins. Indeed, despite the wealth of data on the role of defensins in innate immunity, remarkably little is known about their biosynthesis. Using recombinant prodefensins as "bait" in yeast two-hybrid experiments, we identified SDF2L1 a component of an endoplasmic reticulum (ER) chaperone complex to be a key element in defensin biosynthesis. These preliminary studies suggest that SDF2L1 and its associated proteins of the ER chaperone complex play a role in defensin-maturation. To elucidate the role of this chaperone complex in processing of myeloid ?-defensins we propose the following specific aims. Specific Aim 1: We will investigate the effect of depletion of SDF2L1 and its associated proteins of the ER-chaperone complex on the processing and localization of prodefensins using biochemical and immunogold-electron microscopy methods. Specific Aim 2: We will characterize the interactions of the precursor/mature HNP with the ER chaperone complex by GST-pull down, co-immunoprecipitation and biotin-label transfer studies. Specific Aim 3: We will use SDF2L1 as "bait" in a three-hybrid system to identify proteins whose interaction with SDF2L1 is specifically influenced by prodefensins, thereby characterizing other components of the putative processing complex. The completion of the proposed studies will provide important insights into defensin biosynthesis and regulation, and thus new understanding of this key component of innate immunity. Additionally, these studies will also further our understanding of the protein folding in the ER, a crucial step in formation secretory proteins. PUBLIC HEALTH RELEVANCE: Defensins are host defense peptides that play numerous roles in innate and acquired immunity. This proposal seeks to characterize the complex of proteins that regulates the post-translational processing and localization of human 1-defensins in white blood cells which is critical in understanding the expression of these important effectors of the innate immune system.